ABSTRACT Uterovaginal developmental abnormalities occur in ~7-10% of women and impair reproductive function. Congenital absence of the uterus/vagina, known as Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome, represents the most drastic Mullerian anomaly. Other problems such as unilateral renal agenesis, skeletal, cardiac, deafness, and emotional stress may occur in MRKH. There is a genetic predisposition, and autosomal dominant (AD) and genetic heterogeneity are likely. Many candidate chromosomal regions and genes have been studied, but there is only convincing evidence for mutations in 2 genes (WNT4 and HNF1B) as determined by in vitro analyses and family studies. A critical barrier to diagnose and counsel MRKH women is the lack of understanding of its pathogenesis. We found a new gene?ZNHIT3?by whole exome sequencing (WES), supported by a de novo heterozygous frameshift mutation, two intragenic exon 5 deletions, three whole gene deletions, and two 17q12 deletions including ZNHIT3. ZNHIT3 is zinc finger protein important for post- transcriptional modification, which we showed is expressed in the human uterus, kidney, and heart. Our goal is to understand the molecular basis and pathogenesis of urogenital anomalies by identifying genetic causes of MRKH. Genes and mechanisms identified as a result of this proposal support our translational strategy to identify and understand pathways important in human Mullerian development. Our central hypothesis is that MRKH is AD, and is caused by intragenic mutations and copy number variants (CNVs). Our expected outcomes include the identification of genes causing MRKH and associated anomalies. The impact of our findings include: providing the basis for improved diagnosis, genetic counseling, and prevention of MRKH; and increasing our understanding of normal Mullerian developmental pathways, which are relevant to more common, less severe anomalies. Aim 1: We hypothesize that point mutations, splicing mutations, and CNVs will be present in ZNHIT3, perhaps a hotspot for CNVs and rearrangements. We will perform RT-PCR and Sanger DNA sequencing on MRKH patient lymphoblast RNA to identify multiple types of mutations in one assay. We will determine in vitro effects of human ZNHIT3 variants and generate a Znhit3 knockout mouse model. Aim 2: To test the hypothesis that MRKH is AD, we will perform WES on 43 quads and 26 trios and filter with standard methods, and use Cartagenia, PhenoDB, and pVAAST to determine MRKH inheritance. We will perform whole genome sequencing (WGS) to identify variants that would be missed by WES in well characterized quad families. Identified variants will be confirmed by Sanger sequencing. Aim 3: Perform a comprehensive analysis of MRKH families to determine the frequency and spectrum of variants in known and suspected MRKH genes by RT-PCR from lymphoblast RNA and Sanger sequencing. Our strong preliminary data, large family cohort, innovative approach, and interdisciplinary research team will instigate significant advances in human Mullerian development as outlined by the Fertility & Infertility Branch of the NICHD.